Significant research effort has been directed toward the development of techniques to introduce unnatural amino acids into polypeptide chains, either by chemical synthesis (see, for example, Hofman et al., J. Am. Chem. Soc. 88:5914, 1966), semi-synthetic approaches (see, for example, Borras et al., Nature 227:716, 1970; Sealock et al., Biochemistry 8:3703, 1969; Inouye et al., J. Am. Chem. Soc. 101:752, 1979), modification of reactive side-groups in extant polypeptides (see, for example, Neet et al., Proc. Natl. Acad. Sci. USA 56:1606, 1966; Polgar et al., J. Am. Chem. Soc. 88:3153, 1966; Kaiser et al., Science 266:505, 1984; Mayo et al., Science 233:948, 1986), or use of alternatively acylated tRNAs (see, for example, Krieg et al., Proc. Natl. Acad. Sci. USA 83:8604, 1986; Wiedmann et al., Nature 328: 830, 1987; Johnson et al., Biochemistry 15:569, 1976; Baldini et al., Biochemistry 27:7951, 1988; Roesser et al., Biochemistry 25:6361, 1986; Heckler et al., J. Biol. Chem. 258:4492, 1983; Noren et al., Science 244:182, 1989; Ellman et al., Met. Enzymol. 202:301, 1991). Introduction of such unnatural amino acids into proteins allows analysis of protein folding and/or activity, and also allows adjustment of protein characteristics such as solubility, stability, etc.
Unfortunately, most of the techniques available for introducing unnatural amino acids into proteins generate only low protein yields. Furthermore, many techniques can only be utilized in vitro and/or rely or laborious synthetic technologies. Also, those techniques that utilize alternatively acylated tRNAs can typically introduce only a single unnatural amino acid into a given polypeptide chain. There remains a need for the development of more generally applicable systems for introducing unnatural amino acids into proteins. Preferably, such systems should allow unnatural amino acids to be incorporated into growing polypeptide chains in vivo. Alternatively or additionally, such systems should be able to introduce multiple unnatural amino acids into a single protein.
Significant research effort has also been directed at developing techniques and reagents for the treatment or cure of various human genetic diseases. There remains a need for the development of improved systems.